Effect of Low-NaCl Medium on the Envelope Glycoproteins of Sindbis Virus

Transcription

1 JOURNAL OF VIROLOGY, Mar. 1978, p X/78/ $02.00/0 Copyright i 1978 American Society for Microbiology Vol. 25, No. 3 Printed in U.S.A. Effect of Low-NaCl Medium on the Envelope Glycoproteins of Sindbis Virus JOSEPH W. BELL, JR.,t ROBERT F. GARRY, AND MARILYNN R. F. WAITE* Department of Microbiology, The University of Texas, Austin, Texas Received for publication 16 May 1977 Lowering the NaCl concentration of the medium inhibits the release of Sindbis virus from infected chick cells at a stage after the nucleocapsids have bound to the membranes of the infected cells. The failure of trypsin treatment to release the inhibited virus and the ratio of the proteins in the inhibited cells make it seem likely that the inhibited virus is all intracellular. Experiments using antisera specific for El and E2, the envelope glycoproteins of Sindbis, suggest that the inhibitory effect of low-salt medium is mediated through an effect on E2. Lactoperoxidase radioiodination experiments indicate that, even when cleaved from PE2, E2 is not exposed on the surface of low-nacl-treated chick cells. When chicken embryo (CE) cell cultures infected with Sindbis virus, a small, enveloped RNA virus, are placed in a medium in which the NaCl concentration has been lowered from to M, maturation of the infectious particles is inhibited 95 to 99% (17, 18). Returning the cells to normal medium reverses the inhibition. The number of virus particles produced within a few minutes after the shift to normal medium approaches that released by control cultures throughout the entire incubation period. Treatment of cells incubated in lowsalt medium with anti-sindbis serum prevents virus release if the cells are shifted to normal medium, indicating that some membrane modification occurs in the inhibitory medium. Inhibited cells disrupted in low-salt medium and shifted to normal medium release as many virions as intact cells, evidence that morphogenesis is halted after nucleocapsid binding to the cell membrane occurs (17, 18). Electron microscopy of low-salt-inhibited, Sindbis-infected cells revealed that at least one-half of the unreleased virions were arrested in early stages of budding. The remainder of the virus appeared to be mature and precipitated on the surface of the cell (16). Since preparation of the cells for electron microscopy involved fixation with glutaraldehyde or formaldehyde, procedures which could possibly cause nearly mature virus to finish budding, we wished to determine the extent of virus maturation in low-salt-treated cells. Mild trypsin treatment of RNA tumor virus-infected cells released mature RNA tumor viruses (1), but similar treatment of the low-salt-inhibited, Sindbis-infected cells failed to release any Sindbis. t Present address: Department of Microbiology, Saint Francis Hospital, Wichita, KS This led us to further experiments to determine the site of inhibition by the low-salt medium. Our current data suggest that all the inhibited virions are intracellular, and that the low-salt medium affects the ability of the envelope glycoprotein E2 to penetrate the cell membrane. MATERIALS AND METHODS Viruses, cells, and media. The growth and quantitation of Sindbis virus in CE cell cultures have been described previously (7). Normal medium is a term used to describe Eagle medium with Hanks salts. It has an NaCl concentration of M. Low-salt medium, which has been described previously (16-18), has an NaCl concentration of M. Hanks balanced salt solution is used where indicated, and low balanced salt solution follows the same formula, except that 50% of the NaCl has been omitted. Radioactive labeling. To label cell-associated virus proteins with an amino acid label, CE cells were pretreated for 1 h in leucine- or methionine-free normal medium containing actinomycin D (5 jig/ml). Cultures were infected at room temperature at a multiplicity of 10 PFU/cell. After 1 h for virus adsorption, the inocula were removed, and leucine- or methioninefree medium containing the indicated amount of NaCl and actinomycin D (5 ug/ml) was added. At the times specified in the figure legends, this was replaced with identical medium supplemented with appropriate isotope. To label cell-associated protein, either 10 pci of [3H]leucine (Amersham/Searle; specific activity, 50 Ci/mmol) or 20 pci of [3S]methionine (Amersham/Searle; specific activity, 380 Ci/mmol) per ml was added to the culture. Incubation was continued for the amount of time specified in the figure legends, and further treatment was as described. Chase medium for the [3S]methionine experiment contained 0.38 M NaCl and 100 times as much unlabeled methionine as normal medium. To prepare [3H]leucine-labeled, purified virus, cultures were treated as described above, except that the cultures were labeled from 3 to 9 h after infection 764

2 VoL. 25, 1978 LOW-NaCl MEDIUM AND SINDBIS ENVELOPE PROTEINS 765 with [3H]leucine (50,uCi/ml). The virus was purified from the culture medium as described previously (10). Lactoperoxidase radioiodination was carried out by a modification of the method of Sefton et al. (14). Sindbis-infected CE cells were incubated in low-salt or normal medium and labeled with [3H]leucine between 5 and 6 h after infection as described above. The cultures were washed three ttmes in low-salt solution (0.08 M NaCl-0.01 M Tris [ph 7.5]-10,M glucose) or normal salt solution (0.14 M NaCl-0.01 M Tris [ph 7.5]-10,uM glucose). lodination was carried out in 2 ml of one of the above solutions, which also contained lactoperoxidase (6 Ig; Sigma), Nal (2 nmol), and NaliI (100,uCi/ml; Amersham/Searle). Glucose oxidase (0.025 U; Worthington) was added to start the reaction. After 10 min, the solutions were removed and the cultures were prepared for polyacrylamide gel electrophoresis. [3H]leucine-labeled, purified Sindbis was suspended in 1 ml of the low- or normal-salt solution used for the radioiodination reaction. Samples of the same virus preparation were disrupted with 1% sodium dodecyl sulfate and dialyzed against these buffers. To 1 ml of virus preparation, lactoperoxidase (3 jig), Nal (1 nmol), and NaliI (100 uci/ml) were added. The reactions were started by the addition of H202 (50 ymol). Additional H202 (50 jtmol) was added after 5 min. The reactions were terminated after 10 min by the addition of an equal volume of double-strength digestion mix, and the proteins were separated by polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis. Cell cultures prepared for electrophoresis were dissolved by direct addition of 4% sodium dodecyl sulfate in M Tris (ph 6.8). After the addition of 5%,B-mercaptoethanol, the lysates were heated at 1000C for 2 min and subjected to electrophoresis. Slab gels were prepared by the method of Laemmli (8) in an apparatus (Hoeffer Scientific) similar to that described by Reid and Bieleski (9). The gels were dried on a commercial gel drier (Hoeffer Scientific). [ns]methionine-labeled preparations were exposed to Chronex 2 DC X-ray film (E. I. du Pont de Nemours & Co.) for 2 to 4 days. The developed autoradiograms were quantitated by densitometry, using methods and equipment (Beckman Analytrol) similar to those described by DeRosier and co-workers (5). If the preparations were labeled with [3H]leucine and 111, the tracks followed by the different preparations were separated and cut into 1- mm slices. These slices were placed in test tubes, and the amount of "1I per slice was determined by counting in a gamma counter. The slices were then transferred to scintillation vials containing 0.2 ml of water. After overnight incubation, Aquasol was added, and the amount of [3H]leucine present was determined in a liquid scintillation counter. Antiserum treatments. Antisera to the purified El and E2 glycoproteins of Sindbis virus were kindly provided for these experiments by Joel Dalrymple. Characterization of these antisera has been described previously (4). All antisera were diluted 1:10 in the appropriate medium before use. RESULTS Protease treatments. Previous results suggested that many of the Sindbis virions whose release was inhibited by low-salt medium were mature and precipitated on the cell surface (16). In an attempt to release the ostensibly mature virions (1), we treated the cells with various concentrations of trypsin in Hanks balanced salt solution containing a decreased amount of NaCl. No significant amount of infectious virus was released, although the concentrations of trypsin used were shown not to inactivate free Sindbis virus (Fig. 1). Other experiments using [3H]uridine-labeled, low-salt-treated cells demonstrated that neither trypsin nor chymotrypsin released radioactively labeled, noninfectious virions (not shown). These results led us to further examine the effect of low-salt medium on the release of Sindbis virus from CE cell cultures. 0 s TRYPSIN CONCENTRATION (mg/ml) FIG. 1. Effect of trypsin on virus release from lowsalt-treated cells. CE cultures infected with Sindbis were incubated in low-salt or normal medium for 8 h. They were then washed with Hanks balanced salt solution (HBSS) or a similar solution containing only O.O69MNaCI (LBSS). The cultures were then exposed to the indicated concentration of trypsin diluted in the appropriate balanced salt solution. After 20 min at 37 C, the solutions were harvested, mixed with an equal volume of normal medium containing 20% serum, and centrifuged at 12,000 x g in a refrigerated centrifuge for 15 min to remove cell debris. The samples were kept chilled until adsorption to the monolayers used for virus titration was complete, less than 3 h. The amount of virus released from normal cultures by trypsin was plotted as the percentage of the amount released into normal HBSS (2.8 x 167 PFU/ml; 0). The amount of virus released by inhibited cultures into trypsin-containing LBSS was plotted in two ways: as a percentage of that released by a culture incubated in LBSS alone (1.2 x 106 PFU/ml; U) and as a percentage of that released by an inhibited culture shifted into nonnal HBSS (109 PFU/ml; UW-). To control for protease degradation of Sindbis, virus (10' PFU/ml) suspended in LBSS (E) or HBSS (0) was also treated with the same protease solutions.

3 766 BELL, GARRY, AND WAITE Virus-specified proteins. To determine whether the virus in the inhibited cells was mature or not, we examined the proteins in the inhibited cells. Sindbis virions contain two envelope glycoproteins, El and E2 (11). Little E2 is found within the infected cell since it is cut from a slightly larger precursor, PE2, as the virion matures (13). We expected to find either that the cells contained a large excess of E2, indicating that many mature virions were associated with them, or that they had an excess of PE2 and no E2, indicating that PE2 cleavage had been inhibited. This is the case in cells infected with Sindbis mutants with temperature-sensitive defects in the envelope glycoproteins; no virus is released, and PE2 accumulates (3, 6, 7). To our surprise, the relative amounts of virus-specified proteins associated with the inhibited cells did not differ greatly from the control values, although the PE2/E2 ratio was consistently somewhat higher than normal (Fig. z L/) > cc z cc 0m UL) 4 CD S8- CEF- LOW- PULSE SB- CEF- NORMAL-PULSE SS - CEF - LOW CHASE SS - CEF - NORMAL- CHASE PE2 El E2 C DISTANCE MIGRATED (mm) FIG. 2. Slab gel electrophoresis of virus-specified proteins made in low-salt and normal medium. Cultures pretreated with actinomycin D (5 pg/ml) and infected with Sindbis virus (SB) were incubated for 6 h in methionine-free low-salt or normal medium. Two cultures in each medium were then labeled with p3sjmethionine (20 pci/ml) for I h. At the end of the labeling period, one culture from each group was prepared for electrophoresis, and the other was incubated in normal chase mediumn, containing 100 tunes the normal amount of unlabeled methionine, for h and then prepared for ekectrophoresis. Autoradiograms of the gels were scanned on a Beckman Analytrol densitometer. The arrows indicate the positions of the virus-specified proteins PE2 (the precursor to E2), El and E2, the envelope glycoproteins, and C, the capsid protein. CEF, Chicken embryo fibroblasts. J. VIROL. TABLE 1. Ratios of virus-specifiedproteins in Sindbis-infected CE cells incubated in media of low and normal NaCI concentrationa Protein ratio Incubation medium E2/E1 PE2/E1 PE2/E2 Normal... Low salt a The reas under the varous peaks shown in Fig. 2 were compared. 2, Table 1). These results suggest that the unreleased virus is not mature, and also indicate that, although some PE2 cleavage does take place in low-salt medium, the remainder, which would result in mature virus, does not occur. Antiserum treatment. To explain the results presented above, we postulated that one of the envelope glycoproteins was affected by the low-salt medium and could not assume the proper configuration to promote virus release. It has been shown previously that anti-sindbis serum, containing antibodies to both El and E2, neutralizes Sindbis infectivity equally effectively in media of low or normal NaCl concentration, and that treatment of low-salt-inhibited cells with anti-sindbis serum prevents virus release if the cells are subsequently shifted to normal medium (18). Since antibody does not enter living cells, these experiments demonstrated that the plasma membrane is modified in cells incubated in low-salt medium, but did not indicate to what extent it had taken place. At the time these experiments were done, the presence of two envelope glycoproteins in Sindbis virus had not been demonstrated. Recently, antisera specific for purified El and E2 became available, and we used them to show that both El and E2 could be detected on the surface of Sindbisinfected cells incubated in normal medium (2). To determine whether this was also the case in low-salt-inhibited cells, we examined the ability of these cells to interact with the specific antisera. All sera were diluted 1:10 in the appropriate medium before use. Inhibited and control cells were treated with antiserum in medium of the proper salt concentration, washed in fresh medium of the same NaCl concentration, and then incubated for 30 min in normal medium. The virus released into the normal medium was titrated (Table 2). As reported previously (18), anti-sindbis serum prevented the production of virus from both normal and inhibited cells. Anti- El and anti-e2 sera both prevented the release of infectious virus from control cells, and anti- El treatment of low-salt-inhibited cells had the same effect. Anti-E2 serum, however, had little effect on the amount of virus released after reversal of the inhibition due to the low-salt

4 VOL. 25, 1978 TABLE 2. Effect of antisera on release of infectious virus from Sindbis-infected ceus incubated in lowsalt or normal medium Vimus released into nor- Growth medium' Antiserumb mal medium after antiserum treatmente Normal Control 3.4 x 107 Anti-SBd 5.7 x 104 Anti-El 5.4 x 104 Anti-E2 5.7 x 104 Low salt Control 3.0 x 109 Anti-SB 6.6 x 107 Anti-El 1.9 x 107 Anti-E2 1.1 x 109 a Cultures were incubated for 8 h after infection in low-salt or normal medium. b Cultures were then incubated for an additional 30 min in similar medium containing a 1:10 dilution of the indicated antiserum. After 30 min at 37 C, the antisera were removed, and the cultures were washed five times with fresh medium of the same salt concentration. cnormal medium was added to all cultures, and incubation was continued for 30 min. The virus released during this 30-min period was titrated. d SB, Sindbis virus. medium. Since anti-e2 serum is known to inhibit the infectivity of the virus (4), recognizable E2 antigenicity must have been absent from the surface of the low-salt-inhibited cells. Anti-E2 serum is not just a weak antiserum to El. Anti-El serum does inhibit virus infectivity slightly (75% at a 1:12 dilution). This could, however, be due to steric hindrance, and anti- E2 serum is a much more potent inhibitor of Sindbis infectivity (75% at a 1:178 dilution; data not shown). Bell and Waite (2), using temperature-sensitive mutants of Sindbis defective in El and E2, have shown that anti-el and anti- E2 react with different viral determinants. Thus, even if anti-el serum does contain some components capable of cross-reacting with E2, the validity of the experiments is not impaired. Radioiodination studies. The results of the antiserum experiments suggest that low-nacl medium inhibits virus release by preventing E2 from penetrating the membrane, even after it has been cleaved from PE2. Since E2 is the major protein species on the extemal surface of the plasma membrane of infected cells that can be radioiodinated with lactoperoxidase (14), this hypothesis could be easily tested. [3H]leucine was used to label the virus-specified proteins made in Sindbis-infected CE cells incubated in low-salt or normal medium. The surface proteins of these cells were then labeled with 1"I, using a modification of the procedure of Sefton et al. (14). Cell extracts were analyzed by sodium do- LOW-NaCl MEDIUM AND SINDBIS ENVELOPE PROTEINS 2 C z 0 i IL T 7Cr DISTANCE MIGRATED (mm) 767 FIG. 3. Lactoperoxidase radioiodination of Sindbis (SB)-infected cells incubated under low-salt and normal conditions. CE cells, infected with Sindbis, were incubated in low-salt or normal medium, and the virus-specified proteins were labeled with ph] leucine as described in the text. The cultures were washed with low-salt solution or normal salt solution, and the surface proteins were labeled with III, using the lactoperoxidase reaction described in the text. The reactions were terminated by preparing the cultures for polyacrylamide gel electrophoresis. After electrophoresis, the gels were dried, the tracks were separated, and the gels of the individual samples were cut into 1-mm segments. These were counted in a gamma counter to determine the location of the 125I-labeled species, and then Aquasol was added and they were counted in a liquid scintillation counter to determine the location of the f:hlleucinelabeled viral proteins. Symbols: (0) counts in IHJ leucine; (0) 125I. CEF, Chicken embryo fibroblasts. decyl sulfate-polyacrylamide gel electrophoresis on slab gels. The profiles (Fig. 3) indicated that E2 was not iodinated in the low-salt-treated CE cultures, although it was made in approximately normal amounts. To ascertain that E2 would have been iodinated normally in the low-salt solution, if it has been exposed on the surface of the plasma membrane, similar experiments were carried out using purified Sindbis virus. Four different conditions were used. Intact virus was radioiodinated in normal salt solution and in a solution containing a reduced amount of NaCl. Two other samples were disrupted with sodium dodecyl sulfate and dialyzed against the low- and normal-salt solutions before lactoperoxidase radioiodination. The four samples were analyzed by polyacrylamide gel electrophoresis to determine the lo- 2 2 u 4 cr C,zw w A I

5 768 BELL, GARRY, AND WAITE cation of the [3H]leucine- and '25I-labeled proteins. Lactoperoxidase radioiodination of the E2 of intact virions proceeds at least as efficiently in low-nacl solution as in the normal one (Fig. 4). In agreement with the results of Sefton et al. (14), our data show that disruption of the virion allows iodination of the capsid protein and more extensive iodination of El. Again, the iodination reaction took place normally at both NaCl concentrations. DISCUSSION We showed previously that treatment of Sindbis-infected CE cells with medium of lowered 2 I E E2 c El E2 C A SB-NORMAL B SB-LOW C SDS-SB-NORMAL SDS-SBLOW DISTANCE MIGRATED (mm) FIG. 4. Lactoperoxidase radioiodination of Sindbis virus (SB) under low-salt and normal conditions. Purified, P3Hileucine-labeled Sindbis virus was divided into four samples. Two were treated with sodium dodecyl sulfate (SDS) to disrupt the virus and then dialyzed against the low- and normal-salt solutions used to carry out the reactions. The others were suspended intact in these salt solutions. Lactoperoxidase was used to label the cultures with 125I. The reactions were terminated by the addition of double-strength digestion mix, and the preparations were subjected to slab gelpolyacrylamide gel electrophoresis. The tracks were separated, and the gels were cut into 1-mm slices and counted to determine the location ofthe fhlleucine- and III-labeled viral proteins. The arrows indicate the position of the 19H]leucine-labeled viral structural proteins. The disrupted preparations contained approximately twice as much as the undisrupted ones, so the extent of label ofe2 is comparable. J. VIROL. NaCl concentration inhibits virus release at a very late stage, after the nucleocapsid has bound to the plasma membrane and after the membrane has been modified by the insertion of at least some virus-specified protein (17, 18). Electron microscopic observation of fixed, inhibited CE cells suggested that some of the inhibited virus was mature and precipitated on the outside of the cells, whereas the remainder was arrested at a terminal stage in budding (16). The failure of proteases to release the inhibited virions caused us to re-examine the low-salt phenomenon. The results presented in this paper suggest that the apparently mature Sindbis on the surface of the inhibited cells may have been an artefact of fixation. Polyacrylamide gel electrophoresis profiles of the virus-specified proteins in the inhibited cells did not reveal a great excess of E2, as would be expected if mature virus were on the exterior of the cell, nor did the protein pattern suggest that low-salt medium inhibited the cleavage of PE2 to E2. Experiments using antisera specific for purified El and E2 and lactoperoxidase radioiodination studies indicate that the E2 present in the low-nacl-treated cells is not exposed on the surface of the cell membrane. These results indicate that the inhibition of Sindbis virus release from cells incubated in low- NaCl medium is more complex than was previously suspected. One explanation for the results presented here is compatible with the hypothesis that much of the unreleased Sindbis is mature but precipitated on the surface of the plasma membrane in low-salt medium. In Semliki Forest virus, PE2 is cleaved to give E2 and E3. E3 remains with the virions. E3 is presumably released into the medium of Sindbis-infected cells because it can be found neither in the virus nor in the infected cells. If it were not released, it could possibly block the radioiodination of E2. Since E3 is heavily glycosylated (13), it is easily labeled by tritiated sodium borohydride after exposure to neuraminidase and galactose oxidase. We have been unable to demonstrate the presence of a protein with the mobility expected of E3 in amino acid-labeled or sodium borohydride-treated cells in low-salt medium. Additional glycosylation of E2 occurs after it is cleaved from PE2 but before the virion is released from the cell (13). This glycosylation could be inhibited by the low-salt medium, preventing virus release. The mechanism seems unlikely, however, since glycosylation is an energy-requiring process, and exposure of low-saltinhibited cells to cyanide, fluoride, azide, and iodoacetate does not prevent virus release when the cultures are shifted to normal medium in the continuous presence of the drug (18).

6 VOL. 25, 1978 LOW-NaCI MEDIUM AND SINDBIS ENVELOPE PROTEINS 769 The hypothesis that we favor is that lowering the ionic strength of the medium could alter the membrane fluidity. The capsid could still attach, and a small amount of PE2 could be cleaved to give E2. If the change in fluidity were to prevent E2 from penetrating to the outside surface of the membrane, it could prevent the wave of PE2 to E2 cleavage from traveling around the virion, thereby preventing virus maturation. The lipid composition of the membrane could play an important role in determining how the Sindbisinfected cells will respond to low-nacl medium. We have recently discovered that, in some lines of BHK cells, Sindbis release is susceptible to inhibition by medium of lowered NaCl concentration, whereas in others it is resistant. We are currently comparing these cell lines to determine whether a correlation can be made between response to low-salt medium and lipid composition. ACKNOWLEDGMENTS We thank Joel Dalrymple for providing the antisera to purified El and E2 and Ari Helenius for helpful discussion and suggestions. Ronald M. Smith provided excellent technical asnce. This research was supported by a grant from the University Research Institute of The University of Texas at Austin LITERATURE CrT 1. Allen, D. W Avian myeloblastosis virus (AMV) biosynthesis in chick embryo cells: studies of the particles released by trypsin from the infected cells. Virology 30: Bell, J. W., Jr., and KL R. F. Waite Envelope antigens of Sindbis virus in cells infected with temperature-sensitive mutants. J. Virol. 21: Bracha, K, and KL J. Schleger Defects in RNA' temperature-sensitive mutants of Sindbis virus and evidence for a complex of PE2-E1 viral glycoproteins. Virology 74: Dahrymple, J. K., S. Schlesinger, and P. K. Rusell Antigenic characterization of two Sindbis envelope glycoproteins separated by isoelectric focusing. Virology 69: DeRosier, P. J., P. Munk, and D. J. Cox Automatic measurement of interference photographs from the ultracentrifuge. Anal. Biochem. 50: Jones, K. J., R. K. Scupham, J. A. Pfeil, K. Wan, B. P. Sagik, and H. R. Bose Interaction of Sindbis virus glycoproteins during morphogenesis. J. Virol. 21: Jones, K. J., AL R. F. Waite, and H. R. Bose Cleavage of a viral envelope precursor during the morphogenesis of Sindbis virus. J. Virol. 13: Laemmli, V. K Cleavage of structural proteins dunng the assembly of the head of bacteriophage T4. Nature (London) 227: Reid, M S., and R. L. Bieleski A simple apparatus for vertical flat sheet polyacrylamide gel electrophoresis. Anal. Biochem. 22: Scheele, C. IL, and E. R. Pfefferkorn Kinetics of incorporation of structural proteins into Sindbis virions. J. Virol. 3: Schlesinger, IL J., S. Schlesinger, and B. W. Burge Identification of a second glycoprotein in Sindbis virus. Virology 47: Schlesinger, S., and ML J. Schlesinger Formation of Sindbis virus proteins: identification of a precursor for one of the envelope proteins. J. Virol. 10: Sefton, B. K, and K. Keegstra Glycoproteins of Sindbis virus: preliminary characterization of the oligosaccharides. J. Virol. 14: Sefton, B. K, G. G. Wickus, and B. W. Burge Enzymatic iodination of Sindbis virus proteins. J. Virol. 11: Vyazov, S. O., A. D. Altstein, and V. M. Zhdanov Virions released from the surface of leukovirusproducing cells. Arch. Gesamte Virusforsch. 46: Waite, KL R. F., D. T. Brown, and E. R. Pfefferkorn Inhibition of Sindbis virus release by media of lowered ionic strength: an electron microscope study. J. Virol. 10: Waite, KL R. F., and E. R. Pfefferkorn The effect of altered osmotic presre on the growth of Sindbis virus. J. Virol. 2: Waite, K R. F., and E. R. Pfefferkorn Inhibition of Sindbis virus production by media of low ionic strength: intracellular events and requirements for reversal. J. Virol. 5:60-71.